1. Field of the Invention
The present invention relates to a nucleotide primer set and a detection probe for detecting a genotype in a single-nucleotide polymorphism of a methylene tetrahydrofolate reductase (MTHFR) gene.
2. Description of the Related Art
Methylene tetrahydrofolate reductase (MTHFR) is an enzyme involved in the metabolism of folic acid. It is essential for normal metabolism of homocysteine. The gene coding MTHFR has single-nucleotide polymorphisms C677T and A1298C. Mutation on the MTHFR gene alters its amino acid configuration and lowers its activity. Mutation on the MTHFR gene may induce hyperhomosysteinemia and is reported to increase the risk factor to arteriosclerotic diseases and colon cancer. In addition, correlation between the possibility of occurrence of side effects of methotrexate, which is an antirheumatic, and MTHFR gene polymorphism has been also reported.
Accordingly, it is valuable to analyze mutation of the MTHFR gene for prevention of medicinal side effects. It is also possible to perform pharmaceutical administration and treatment tailor-made to the patient, by determining the genotype of the MTHFR gene.
The single-nucleotide polymorphism is generally detected by amplifying a target nucleotide with the polymerase chain reaction (PCR) method and detecting wild-type and variant amplification products with a specific probe (see, the reference Jain K. K., Application of Amplicip. CYP450, Mol Diagn. 9, 119-27 [2005]). However, the PCR method has disadvantages such as complicated procedure of pretreatment including nucleotide extraction, demand for a complex temperature-regulating device such as a thermal cycler, and a longer reaction period of two hours or more. Amplification products by the PCR method are double-stranded chains, and thus, there is a problem in that the complementary chain degrades the detection sensitivity, while functioning as a competitor to the probe during detection. Various methods of converting the amplification product into a single-stranded chain, for example, by decomposing or separating a complementary chain by using an enzyme or magnetic beads, have been studied, but these methods also have a problem in that the operation is complicated and expensive.